Water-wettable chromatographic media for solid phase extraction

ABSTRACT

A method for removing an organic solute from a solution, comprising contacting the solution with a polymer formed by copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers, whereby the solute is adsorbed onto the polymer. The solution can comprise a polar solvent such as a polar organic solvent or water or an aqueous buffer. The hydrophobic monomer can be, for example, divinylbenzene. The hydrophilic monomer can be, for example, a heterocyclic monomer, such as a vinylpyridine or N-vinylpyrrolidone.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/643,094, filed Aug. 21, 2000, which is a continuation application ofU.S. application Ser. No. 09/374,945 filed on Aug. 16, 1999 ( U.S. Pat.No. 6,106,721; issue date Aug. 22, 2000), which is a continuationapplication of U.S. Ser. No. 09/216,047, filed on Dec. 18, 1998 (U.S.Pat. No. 5,976,367; issue date Nov. 2, 1999), which is a divisionalapplication of U.S. Ser. No. 08/634,710 filed on Apr. 18, 1996 (U.S.Pat. No. 5,882,521; issue date Mar. 16, 1999). The teachings of each ofthese referenced applications are expressly incorporated herein byreference in their entirety.

BACKGROUND OF THE INVENTION

[0002] Solid phase extraction is a chromatographic technique of frequentuse in the preparation of samples for quantitative analysis, forexample, via high performance liquid chromatography (HPLC) or gaschromatography (GC) (McDonald and Bouvier, eds. Solid Phase ExtractionApplications Guide and Bibliography, sixth edition, Milford, Mass.:Waters (1995)). Solid phase extraction can be used to separate acomponent of interest in a complex solution from potentially interferingmatrix elements and to concentrate the analyte to levels amenable todetection and measurement. Thus, solid phase extraction is of use in theanalysis of environmental samples, where, for example, various solublecomponents of soils may interfere with the analysis of trace organicmaterials. Solid phase extraction is also of importance in the analysisof pharmaceutical agents or metabolites in blood plasma, which requiresthe prior removal of plasma proteins and other matrix constituents whichmay interfere with the analysis.

[0003] Solid phase extraction of an aqueous solution is typicallyperformed by passing the solution through a single-use cartridgecontaining a chromatographic sorbent. The most commonly used sorbentsconsist of porous silica particles that have been functionalized ontheir surface with hydrophobic octyl (C₈) and octadecyl (C₁₈) functionalgroups. Prior to use, such sorbents must be wetted with a water-misciblepolar organic solvent to solvate the alkyl chains. This increases thecontact of these chains with the aqueous phase, increasing the sorbentsurface area available to solutes and, therefore, retention of solutes.Such sorbents which are not pre-wetted or have dried out display poorsolute retention, and, thus, inadequate separation of solutioncomponents.

[0004] The requirement that the sorbent remain wetted during theextraction procedure complicates solid phase extractions andsubstantially slows sample analysis. For example, solid phase extractioncartridges, in general, have differing flow rates and must be monitoredindividually to prevent drying out when used on a vacuum manifold, thecurrent state of the art for processing multiple samples. This furthercomplicates the development of instruments for automated solid phaseextraction, which often incorporate elaborate safeguards to preventdrying out of the sorbent.

[0005] Thus, there is need for a solid phase extraction method whichutilizes a sorbent that does not require wetting with an organic solventor that stays wetted even if the bulk of the wetting solvent is removedduring use on a vacuum manifold. Such a method would enable more rapidsample preparation for quantitative analysis, particularly for multiplesamples, and allow the development of less expensive and simpler methodsfor automated solid phase extraction.

SUMMARY OF THE INVENTION

[0006] The present invention relates to a method for removing an organicsolute from a solution. The method comprises contacting the solutionwith a water-wettable polymer formed by copolymerizing one or morehydrophobic monomers and one or more hydrophilic monomers, whereby thesolute is adsorbed onto the polymer. The solution can comprise a polarsolvent such as a polar organic solvent, a water/organic mixture or,preferably, water or an aqueous solution, such as an aqueous buffer,acid, base or salt solution.

[0007] The hydrophobic monomer can comprise a hydrophobic moiety.Suitable hydrophobic moieties include, but are not limited to phenyl,phenylene and C₂-C₁₈-alkyl groups. Suitable hydrophobic monomers includedivinylbenzene and styrene.

[0008] The hydrophilic monomer can comprise a hydrophilic moiety. In oneembodiment the hydrophilic moiety is a saturated, unsaturated oraromatic heterocyclic group, such as a pyrrolidonyl group or a pyridylgroup. In another embodiment, the hydrophilic moiety is an ether group.Suitable hydrophilic monomers are, for example, N-vinylpyrrolidone,2-vinylpyridine, 3-vinylpyridine, 4-vinylpyridine and ethylene oxide.

[0009] In one embodiment of the method, the polymer is apoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer which comprisesgreater than about 12 mole percent N-vinylpyrrolidone. In a preferredembodiment, the copolymer comprises from about 15 mole percent to about30 mole percent N-vinylpyrrolidone.

[0010] The present invention further includes a method for forming asolution, containing a solute, which is suitable for quantitativeanalysis. In one embodiment, the method comprises contacting a firstsolution including the solute with a water-wettable polymer formed bycopolymerizing at least one hydrophobic monomer and at least onehydrophilic monomer, whereby the solute is adsorbed onto the polymer.This is followed by washing the polymer with a suitable solvent ormixture of solvents, so that the solute is desorbed from the polymer,thereby forming a second solution including the solute. This secondsolution is suitable for quantitative analysis.

[0011] In another embodiment, the invention provides a method forforming a solution comprising a polar organic solute which is suitablefor quantitative analysis. The method comprises contacting a solutionwhich includes the polar organic solute and at least one additionalsolute of lesser polarity with a water-wettable polymer formed bycopolymerizing at least one hydrophobic monomer and at least onehydrophilic monomer, whereby the additional solute is adsorbed onto thepolymer and the polar solute remains in the aqueous phase. The resultingaqueous phase is, thus, a solution of the polar organic solute which issuitable for quantitative analysis.

[0012] The present invention further includes a solid phase extractioncartridge comprising an open-ended container and a polymer packed withinthe container. The solid phase extraction cartridge can, optionally,further comprise a porous retaining means, such as a frit. The polymeris formed by copolymerizing at least one hydrophobic monomer and atleast one hydrophilic monomer. Suitable polymers includepoly(divinylbenzene-co-N-vinylpyrrolidone) copolymers which compriseabout 12 mole percent or more, preferably from about 15 mole percent toabout 30 mole percent, N-vinylpyrrolidone. The solid phase extractioncartridge preferably comprises from about 0.025 g to about 1 g of thepolymer.

[0013] The present invention enables the solid phase extraction of oneor more solutes from an aqueous solution, without prior wetting of thesorbent with an organic solvent. The method is versatile with respect tosolute identity, resulting in extraction of a broad range of solutes ofvarying polarity. A particular advantage of the method is that thesorbent can dry out during the extraction procedure without diminishingthe ability of the sorbent polymer to retain solutes. Thus, the presentinvention provides a simpler method for the preparation of analyticalsamples, decreasing sample preparation time and increasing samplethroughput. The present method is, thus, also more amenable toautomation than currently used methods.

BRIEF DESCRIPTION OF THE DRAWING

[0014] The FIGURE is a schematic, in cross-section, of one embodiment ofthe solid phase extraction cartridge of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention provides a method for solid phaseextraction of aqueous or buffered aqueous solutions which does notrequire that the sorbent be wetted with an excess of organic solventprior to and during the solid phase extraction process. The invention isbased on the discovery that polymers or resins comprising both ahydrophilic monomer and a hydrophobic monomer in a suitable ratio can bewetted by water while maintaining surprisingly effective retention oforganic solutes with a wide range of chromatographic polarities.

[0016] As described in the Exemplification, a relatively small increasein the N-vinylpyrrolidone content of apoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer resulted in adramatic improvement in retention of polar organic solutes underconditions in which the pre-wetted polymer was dried under reducedpressure for several minutes. For example, under these conditions,recovery of acetaminophen from such a copolymer comprising 9 molepercent N-vinylpyrrolidone was 10.4%. Increasing the mole percentN-vinylpyrrolidone in the copolymer to 13 resulted in a 92% recovery ofacetaminophen. Similar results were observed for procainamide,ranitidine, and caffeine. For relatively nonpolar solutes the differencein recovery between the two copolymers was less dramatic.

[0017] The ability of poly(divinylbenzene-co-N-vinylpyrrolidone)copolymers comprising between 13 mole percent and 22 mole percentN-vinylpyrrolidone to retain organic solutes was also compared with thatof octadecyl (C₁₈)-bonded silica gel. As discussed in theExemplification, the C₁₈-bonded silica sorbent showed poor retention ofpolar organic solutes when the sorbent was pre-wetted with an organicsolvent and then dried under reduced pressure prior to extraction. Forexample, this sorbent showed a 2.8% recovery of m-toluamide under theseconditions. In contrast, the poly(divinylbenzene-co-N-vinylpyrrolidone)copolymer comprising 13 mole percent N-vinylpyrrolidone displayed a96.3% recovery of m-toluamide under similar conditions. Overall, theresults demonstrate that a balance between the mutually exclusiveproperties of water-wettability and retention of organic solutes can beachieved in a copolymer which has a suitable ratio of hydrophilicmonomers and hydrophobic monomers.

[0018] In one embodiment, the invention is a method for removing asolute from a solution. The method comprises the step of contacting thesolution with a water-wettable polymer formed by copolymerizing at leastone hydrophobic monomer and at least one hydrophilic monomer, wherebythe solute is adsorbed onto the polymer. The solution can comprisewater, or a mixture of water and a water-miscible polar organic solventsuch as methanol, ethanol, N,N-dimethylformamide, dimethylsulfoxide oracetonitrile. The solution can also comprise a mixture of water or anaqueous buffer and a polar, water-miscible organic solvent. In aparticularly preferred embodiment, the solution is an acidic, basic orneutral aqueous or predominately aqueous, i.e., greater than about 50%water by volume, solution. The solute is preferably an organic compound.

[0019] The solution can be contacted with the polymer in any fashionwhich permits intimate contact of the polymer and the solution, such asa batch or chromatographic process. For example, the solution can beforced through a porous polymer column, disk or plug, or the solutioncan be stirred with the polymer, such as in a batch-stirred reactor. Thesolution can also be added to a polymer-containing well of a microtiterplate. The polymer can take the form of, for example, beads or pellets.The solution is contacted with the polymer for a time period sufficientfor the solute of interest to substantially adsorb onto the polymer.This is typically the time necessary for the solute to equilibratebetween the polymer surface and the solution. The adsorption orpartition of the solute onto the polymer can be partial or complete.

[0020] A preferred polymer for use in the present method iswater-wettable and has the ability to retain a variety of solutes ofvarying polarity. The term water-wettable, as used herein, describes amaterial which is solvated, partially or completely, by water. Thematerial, thus, engages in energetically favorable or attractiveinteractions with water molecules. These interactions increase theamount of surface area of the material which, upon contact with water,is accessible to water molecules, and, hence, to solutes present inaqueous solution.

[0021] The term “monomer”, as used herein, refers to both a moleculecomprising one or more polymerizable functional groups prior topolymerization, and a repeating unit of a polymer. A polymer cancomprise two or more different monomers, in which case it can also bereferred to as a copolymer. The mole percent of a given monomer which acopolymer comprises is the mole fraction, expressed as a percent, of themonomer of interest relative to the total moles of the various (two ormore) monomers which compose the copolymer.

[0022] In one embodiment of the method, the solution is contacted withthe polymer in dry form. In another embodiment the polymer is wettedprior to contacting the solution with the polymer, for example, bytreating the polymer with a polar organic solvent, followed by water oran aqueous buffer.

[0023] The hydrophilic monomer can comprise hydrophilic group. In oneembodiment, the hydrophilic group is a heterocyclic group, for example,a saturated, unsaturated or aromatic heterocyclic group. Suitableexamples include nitrogen-containing heterocyclic groups such aspyrrolidonyl and pyridyl groups. In another embodiment, the hydrophilicmoiety is an ether group. The hydrophilic monomer can be, for example,N-vinylpyrrolidone, 2-vinylpyridine, 3-vinylpyridine, a hydrophobicmoiety, 4-vinylpyridine or ethylene oxide.

[0024] The hydrophobic monomer can comprise, for example, an aromaticcarbocyclic group, such as a phenyl or phenylene group, or an alkylgroup, such as a straight chain or branched C₂-C₁₈-alkyl group. Suitablehydrophobic monomers include, but are not limited to, styrene anddivinylbenzene.

[0025] In a preferred embodiment, the polymer to be contacted with thesolution is a poly(divinylbenzene-co-N-vinylpyrrolidone) copolymer. Thepolymer can comprise about 12 mole percent or more N-vinylpyrrolidone.In a particularly preferred embodiment, the polymer comprises from about15 mole percent to about 30 mole percent N-vinylpyrrolidone.

[0026] The polymer can be in the form of, for example, beads having adiameter in the range from about 5 to about 500 μm, preferably fromabout 20 to about 200 μm. The copolymer, preferably, has a specificsurface area in the range from about 200 to about 800 square meters pergram and pores having a diameter ranging from about 0.5 nm to about 100nm.

[0027] The solution comprising the solute can, optionally, furthercontain one or more additional solutes. In one embodiment, the solutionis an aqueous solution which includes a complex variety of solutes.Solutions of this type include blood plasma, urine, cerebrospinal fluid,synovial fluid and other biological fluids, including extracts oftissues, such as liver tissue, muscle tissue, brain tissue and hearttissue. Such extracts can be aqueous extracts or organic extracts whichhave been dried and subsequently reconstituted in water or in awater/organic mixture.

[0028] The solution can also be ground water, surface water, drinkingwater or an aqueous or organic extract of an environmental sample, suchas a soil sample. The solution can further be a food substance, such asa fruit or vegetable juice or milk or an aqueous or aqueous/organicextract of a food substance, such as a fruit, vegetable, cereal, ormeat.

[0029] The solute can be any organic compound of polarity suitable foradsorption onto the polymer. Such solutes can include, for example,drugs, pesticides, herbicides, toxins and environmental pollutantsresulting from the combustion of fossil fuels or other industrialactivity, such as metal-organic compounds comprising a heavy metal suchas mercury, lead or cadmium. The solutes can also be metabolites ordegradation products of the foregoing materials. The solutes can alsoinclude biomolecules, such as proteins, peptides, hormones,polynucleotides, vitamins, cofactors, metabolites, lipids andcarbohydrates.

[0030] In one embodiment of the method, the polymer is packed asparticles within an open-ended container to form a solid phaseextraction cartridge. The container can be, for example, a cylindricalcontainer or column which is open at both ends so that the solution canenter the container through one end, contact the polymer within thecontainer, and exit the container through the other end. The polymer canbe packed within the container as small particles, such as beads havinga diameter between about 5 μm and about 500 μm, preferably between about20 μm and about 200 μm. The polymer particles can also be packed in thecontainer enmeshed in a porous membrane.

[0031] The container can be formed of any material which is compatible,within the time frame of the extraction process, with the solutions andsolvents to be used in the procedure. Such materials include glass andvarious plastics, such as high density polyethylene and polypropylene.In one embodiment, the container is cylindrical through most of itslength and has a narrow tip at one end. One example of such a containeris a syringe barrel.

[0032] The solid phase extraction cartridge can further comprise aporous retaining means, such as a filter element, or frit, at one orboth ends of the cartridge adjacent to the polymer to retain the polymerwithin the cartridge and to remove undissolved solid materials from thesolution as it flows into the cartridge, while still permitting solutionflow into and out of the cartridge. Such a filter can be formed from,for example, fritted glass or a porous polymer, such as a porous highdensity polyethylene.

[0033] The amount of polymer within the container is limited by thecontainer volume and can range from about 0.001 g to about 50 g, but ispreferably between about 0.025 g and about 1 g. The amount of polymersuitable for a given extraction depends upon the amount of solute to beadsorbed, the available surface area of the polymer and the strength ofthe interaction between the solute and the polymer. This can be readilydetermined by one of ordinary skill in the art.

[0034] The present invention includes a solid phase extraction cartridgeas described above, wherein the polymer is a water-wettable polymerformed by copolymerizing at least one hydrophobic monomer and at leastone hydrophilic monomer. The polymer can be, for example, apoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer comprising about 12mole percent or more N-vinylpyrrolidone. In a preferred embodiment, thecopolymer comprises from about 15 mole percent to about 30 mole percentN-vinylpyrrolidone. The cartridge can be a single use cartridge, whichis used for the treatment of a single sample and then discarded, or itcan be used to treat multiple samples.

[0035] A preferred embodiment of the solid phase extraction cartridge ofthe present invention is illustrated in cross section in the Figure.Container 1 is a syringe barrel which can be formed of moldedpolypropylene and can have a volume ranging from about 1 cm³ to about 50cm³. Water wettable polymer 2 is prepared by the copolymerization ofN-vinylpyrrolidone and divinylbenzene and comprises from about 12 molepercent to about 30 mole percent N-vinylpyrrolidone. Polymer 2 is packedwithin the container as porous beads of diameter between about 20 μm andabout 200 μm. The mass of polymer 2 packed within the container canrange from about 0.025 g to about 10 g, depending upon the volume of thecontainer. Frits 3 and 4 are formed of porous high density polyethylene.

[0036] The solution to be treated is added to the top of the solid phaseextraction cartridge and allowed to flow through the cartridge, bringingthe solute to be adsorbed into contact with the polymer. The solutioncan flow through the cartridge under the force of gravity. Increasedflow rates can be achieved by establishing a pressure difference betweenthe ends of the cartridge. Such a pressure difference can be establishedby attaching a vacuum source to the lower end of the cartridge or byapplying positive pressure to the upper end of the cartridge, forexample, by applying a pressurized gas, such as air or nitrogen, to thetop of the cartridge, or by compressing the air within the cartridgeabove the polymer with a piston or plunger. The flow rate of thesolution through the cartridge can be adjusted by regulating thepressure difference across the cartridge. Suitable solution flow rates,given in terms of the linear velocity of the solution, range up to about14 mm/second, but are preferably in the range from about 0.7 to about3.5 mm/second.

[0037] Another aspect of the present invention is a method for forming asolution of a solute which is suitable for quantitative analysis. In oneembodiment, the solute is of a polarity suitable for adsorption onto thepolymer. The method comprises contacting a first solution which includesthe solute with a polymer formed by copolymerizing at least onehydrophobic monomer and at least one hydrophilic monomer, whereby thesolute is adsorbed onto the polymer. This is followed by washing thepolymer with a suitable, stronger solvent or mixture of solvents,thereby desorbing or eluting the solute from the polymer and forming asecond solution which contains the solute. This second solution issuitable for the quantitative analysis of the solute.

[0038] The solution contacted with the polymer can comprise the soluteof interest in dilute form, for example, at a concentration too low foraccurate quantitation. By adsorbing the solute onto the polymer and thendesorbing the solute with a substantially smaller volume of a less polarsolvent, a solution which includes the solute of interest can beprepared having a substantially higher concentration of the solute ofinterest than that of the original solution. The method also results insolvent exchange, that is, the solute is removed from a first solventand re-dissolved in a second solvent

[0039] The polymer need not be pretreated or wetted prior to contactingthe solution with the polymer. In one embodiment, the polymer is treatedwith a water-miscible organic solvent, followed by water or aqueousbuffer, prior to contacting the solution with the polymer. In anotherembodiment, the solution is contacted with dry polymer, that is, thepolymer is not wetted prior to treatment of the solution.

[0040] The solution contacted with the polymer can comprise a polarsolvent and is preferably predominately, i.e. greater than 50% byvolume, an acidic, basic or neutral aqueous solution or aqueous buffer.The solution can also comprise a water-miscible polar organic solventsuch as methanol, ethanol, acetonitrile, N,N-dimethylformamide, ordimethylsulfoxide, or a mixture of such a solvent and water.

[0041] The solution comprising the solute of interest can furthercomprise one or more additional solutes. In one embodiment, theadditional solute or solutes are more polar than the solute of interest,and, thus, adsorb more weakly to the polymer than the solute ofinterest. Such an additional solute can be desorbed from the polymer bywashing the polymer with a solvent which does not desorb the compound ofinterest, thereby forming a solution of the additional solute or soluteswhich is substantially free of the solute of interest. A suitablesolvent for the desorption of the additional solute will typically besufficiently polar that it does not desorb the compound of interest.

[0042] After desorption of the additional solute or solutes, thecompound of interest can be desorbed by washing the polymer with asuitable, i.e., less polar, solvent. This forms a solution of theorganic solute which is substantially free from more polar solutes andis suitable for the quantitative analysis of the organic solute.

[0043] In one embodiment, the solute of interest adsorbs onto thepolymer, but one or more additional solutes do not. Such an additionalsolute can be, for example, of sufficiently high polarity that it doesnot adsorb onto the polymer. The additional solute can also compriselarge molecules, for example, macromolecules such as proteins, which areunable to pass through the pores within the polymer, and, thus, haveaccess to only a small fraction of the overall polymer surface area.Such molecules are typically retained poorly, if at all, by the polymer.

[0044] In a further embodiment, the additional solute or solutes areless polar than the solute of interest and, thus, adsorb to the polymermore strongly than the compound of interest. The compound of interestcan be weakly to moderately adsorbed or not adsorbed. If adsorbed, thesolute of interest is desorbed from the polymer by washing the polymerwith a solvent of sufficient polarity that it does not desorb theadditional solute or solutes. Thus, the compound of interest can bedesorbed from the polymer without desorbing the other solutes.

[0045] In one embodiment, the additional solute or solutes are alsoanalytes of interest. Thus a series of solutes initially present in asolution can be separated, and solutions of each suitable forquantitative analysis can be formed using the method of the presentinvention. In this case, the solution is contacted with the polymer sothat the solutes adsorb to the polymer. The solutes are then desorbedfrom the polymer in order of decreasing polarity (i.e., most polarsolute first, followed by solutes of successively decreasing polarity)by washing the polymer with a sequence of solvents of decreasingpolarity.

[0046] Polymers, solutions and solutes which are suitable for thismethod include those described above. Solvents which are suitable fordesorbing the solute from the polymer will typically be polarwater-miscible organic solvents, such as alcohols, for example,methanol, ethanol, and isopropanol, acetonitrile, acetone, andtetrahydrofuran, or mixtures of water and these solvents. The desorbingsolvent can also be a nonpolar or moderately polar water-immisciblesolvent such as dichloromethane, diethylether, chloroform, orethylacetate. Mixtures of these solvents are also suitable. Preferredsolvents or solvent mixtures must be determined for each individualcase. A suitable solvent can be determined by one of ordinary skill inthe art without undue experimentation, as is routinely done inchromatographic methods development (McDonald and Bouvier, supra,(1995); Snyder and Kirkland, Introduction to Modern LiquidChromatography, New York: J. Wiley and Sons (1974)).

[0047] The methods of the present invention can be used to preparesolutions of a solute which are suitable for quantitative analysis via avariety of techniques, including high performance liquid chromatography,gas chromatography, gas chromatography/mass spectrometry, andimmunoassay.

[0048] The sorbent polymers used in the methods of the present inventioncan be prepared via standard synthetic methods. For example, apoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer can be synthesizedby copolymerization of divinylbenzene and N-vinylpyrrolidone usingstandard methods of free radical polymerization which are well known inthe art. One method for forming copolymers of this type is disclosed inU.S. Pat. No. 4,382,124, issued to Meitzner et al., the contents ofwhich are incorporated herein by reference. The composition of theresulting copolymer depends upon the starting stoichiometry of the twomonomers and can be readily varied. The composition of the productcopolymer in some cases will not be substantially the same as theproportion of the starting materials, due to differences in reactivityratios among the monomers.

[0049] The invention will now be further and specifically described bythe following example.

EXEMPLIFICATION Materials

[0050] The model solutes procainamide, acetaminophen, m-toluidine,m-toluamide, propranolol, caffeine, and 2,7-dihydroxynaphthalene wereobtained from Aldrich Chemical Company (Milwaukee, Wis.), while doxepin,ranitidine, and betamethasone-17-valerate were purchased from SigmaChemical Company (St. Louis, Mo.). The tC₁₈ bonded silica solid phaseextraction cartridge was obtained from Waters Corporation (Milford,Mass., catalogue no. WAT054960). Apoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer comprising about 9mole percent N-vinylpyrrolidone was obtained from Waters Corporation(Porapak®R). Poly(divinylbenzene) was also obtained from Waters(Styragel®).

Preparation of Poly(Divinylbenzene-co-N-Vinylpyrrolidone) Copolymers

[0051] To a 3000 mL flask was added a solution of 5.0 ghydroxypropylmethylcellulose (Methocel E15, Dow Chemical Co., Midland,Mich.) in 1000 mL water. To this was added a solution of 175 gdivinylbenzene (DVB HP-80, Dow), 102 g N-vinyl-2-pyrrolidone(International Specialty Products), and 1.85 g azobisisobutyronitrile(Vazo 64, Dupont Chemical Co, Wilmington, Del.) in 242 g toluene. Theresulting biphasic mixture was stirred for 30 minutes at roomtemperature using sufficient agitation to form oil droplets of thedesired micron size. The resulting suspension was then heated undermoderate agitation to 70° C. and maintained at this temperature for 20hours. The suspension was then cooled to room temperature, filtered andwashed with methanol. The filter cake was then dried in vacuo for 16hours at 80° C. The composition of the product polymer was determined byelemental analysis.

[0052] Elemental analysis: N: 2.24%; mole percent N-vinylpyrrolidone:20%.

[0053] A series of poly(divinylbenzene-co-N-vinylpyrrolidone) copolymerscomprising about 13, 14, 16, and 22 mole percent N-vinylpyrrolidone wasalso prepared by this method by varying the starting ratio of thedivinylbenzene and N-vinylpyrrolidone monomers.

[0054] A 50 mg amount of each polymer was packed into a 1 cc Sep-PakVac® cartridge container (Waters Corporation) having a polyethylene fritat both the inlet and the outlet of the polymer bed to form a solidphase extraction cartridge.

Method

[0055] Each model compound was dissolved in 20 mM phosphate buffer, pH7, to form a solution having a concentration of 10 μg/mL.

[0056] Solid phase extraction of model solutes

[0057] The solutions of the model solutes were subjected to solid phaseextraction on solid phase extraction cartridges conditioned under twosets of conditions. In both cases the cartridge was attached to a vacuummanifold and treated with 1 mL methanol. The vacuum was set to about 4Hg, to give a methanol flow rate of 1 mL /minute. Under the first set ofconditions (wet conditions), the vacuum was released when the methanollevel reached the top of the sorbent. Under the second set of conditions(“dry conditions”) the sorbent was allowed to dry out under vacuumfollowing conditioning with methanol. As in the first method, thecartridge was treated with 1 mL methanol, at a flow rate, under reducedpressure (4″ Hg), of about 1 mL/minute. When the methanol level reachedthe top of the sorbent, the vacuum was set to 10″ Hg and maintained for10 minutes to dry the polymer bed.

[0058] In both wet and dry cases, 1 mL of the model compound solutionwas applied to the cartridge at a flow rate of 1 mL/minute. A 1 mLportion of 20 mM phosphate buffer, pH 7 was then added at a flow rate of1 mL/minute. A 1 mL portion of methanol was then added at a flow rate of1 mL/minute to desorb and eluate the model compound. To the eluate wasadded an internal standard, and the model compound within the eluent wasquantitated by high performance liquid chromatography.

Results

[0059] The results are summarized in the table below, which lists polarcompounds (procainamide, acetaminophen and ranitidine), moderately polarcompounds (caffeine, m-toluamide, m-toluidine, 2,7-dihydroxynaphthalene,and propranolol) and nonpolar compounds (dipropylphthalate, doxepin andbetamethasone-13-valerate). When the sorbent was poly(divinylbenzene)all compounds except doxepin showed greater than 89% recovery when thesorbent was conditioned under wet conditions. Recovery of doxepin, asshown, was significantly lower because this compound required greaterthan 1 mL methanol for quantitative elution. When the sorbent wastreated under dry conditions, only dipropylphthalate and betamethasonevalerate were recovered in greater than 80% yield, and recovery ofprocainamide, acetaminophen and ranitidine fell below 10%.

[0060] When the sorbent was tC₁₈-bonded silica, each compound tested wasrecovered in high yield (>85%) under wet conditions. Allowing thesorbent to dry out had negligible effect on the recovery ofdipropylphthalate, doxepin and betamethasone valerate, but reduced theyield of caffeine, m-toluidine, m-toluamide, 2,7-dihydroxynaphthaleneand propranolol to about 13% or less.

[0061] When the sorbent was a poly(divinylbenzene-co-N-vinylpyrrolidone)copolymer, recovery of each compound was in the range of about 80-100%when the sorbent was kept wetted. When the copolymer composition was 9mole percent N-vinylpyrrolidone, high recovery of the nonpolar compoundswas noted under both wet and dry conditions. The more polar compoundswere recovered in high yield under wet conditions but in sharply reducedyield under dry conditions. Recovery of these compounds under dryconditions dramatically increased when the N-vinylpyrrolidone componentof the copolymer was increased to about 13 mole percent or greater. Therecovery of these compounds under wet conditions was essentiallyinvariant as the copolymer composition was changed. TABLE Comparison ofSPE recoveries for various model compounds Analyses performed intriplicate. DVB = divinylbenzene, NVP = N-vinylpyrrolidone- % NVP givenas mole percent NVP. Percent Recovery (Average) Poly(DVB- Poly(DVB-Poly(DVB- Poly(DVB- Poly(DVB- Poly(DVB- Poly(DVB- co-NVP) co NVP) coNVP) co-NVP) co NVP) co-NVP) co-NVP) Poly(DVB) 9% NVP 13% NVP 14% NVP16% NVP 20% NVP 20% NVP 22% N IC_(1R) Compound wet dry wet dry wet drywet dry wet dry wet dry wet dry wet dry wet dry Procainamide 95.4 2.5*90.8 4.9* 84.8* 94.2 84* 99.9 98.8 89.1 88.3 92.3 85.7 93.3Acetaminophen 98.0 2.0* 93.8* 10.4* 92.3 97.9 89.4* 104.5 104.4 96.294.8 93.4 96.7 102.2 Ranitidine 95.0 5.4* 89.6 13.4* 99.7 93.5 88.4 98.397.6 86.0 85.2 92.1 76.9 86.6 Caffeine 95.6* 30.8* 101.0 25.6* 96.0 95.798.7 96.8 99.7 97.3 97.7 98.2 99.2 95.4 103.9 1.5* Toluamide 98.5 54.8*101.1 74.4* 96.5 96.3 100.8 96.4 100.0 97.0 97.0 97.3 100.5 96.3 103.72.8* Toluidine 95.6 80.7* 102.8 96.8 97.6 98.0 96.5 93.4 94.0 93.2 95.196.7 91.9 90.3 101.7 13.4* 2,7-Dihydroxy- 99.5 46.5* 103.6 89.6* 96.896.3 95.9 94.2 96.7 95.4 95.7 94.5 94.4 90.0 102.8 0* naphthalenePropranolol 92.3 55.8* 102.1 94.5 94.2 92.4 94.1 95.4 88.5 85.5 88.088.3 98.2 9.3* Dipropylphthalate 91.5 99.2 102.1 101.6 93.1 100.0 89.589.3 88.9 86.0 89.1 98.5 92.2 98.4 Doxepin 47.5⁺ 55.4⁺ 85.3 86.8 77.5⁺78.0 84.1 81.8 84.7 77.5 78.6 79.9 95.1 104.0 Betamethasone-13- 89.183.6 93.3 97.1 85.9⁺ 89.8 92.7 87.0 84.0 85.0 85.9 85.9 86.6 88.3valerate

Equivalents

[0062] Those skilled in the art will recognize or be able to ascertainusing no more than routine experimentation many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed in the scope of the following claims.

What is claimed is:
 1. A solid phase extraction cartridge comprising awater-wettable polymer packed inside an open-ended container, saidpolymer being formed by copolymerizing at least one hydrophobic monomerand at least one hydrophilic monomer having a hydrophilic to hydrophobicmonomer ratio sufficient for the polymer to be water-wettable whilebeing effective to retain organic solutes over a range ofchromatographic polarities and wherein the polymer comprises greaterthan at least 12 mole percent of hydrophilic monomer.
 2. The solid phaseextraction cartridge of claim I wherein the hydrophilic monomercomprises a heterocyclic group.
 3. The solid phase extraction cartridgeof claim 2 wherein the heterocyclic group is a pyrrolidonyl group or apyridyl group.
 4. The solid phase extraction cartridge of claim 3wherein the hydrophilic monomer is selected from the group consisting of2-vinylpyridine, 3-vinylpyridine and 4-vinylpyridine.
 5. The solid phaseextraction cartridge of claim 3 wherein the hydrophilic monomer isN-vinylpyrrolidone.
 6. The solid phase extraction cartridge of claim 1wherein the hydrophobic monomer comprises a phenyl group, a phenylenegroup or a straight chain or branched C₂-C₁₈-alkyl group.
 7. The solidphase extraction cartridge of claim 6 wherein the hydrophobic monomer isstyrene or divinylbenzene.
 8. The solid phase extraction cartridge ofclaim 1 wherein the polymer is apoly(divinylbenzene-co-N-vinylpyrrolidone) copolymer.
 9. The solid phaseextraction cartridge of claim 8 wherein the polymer comprises about 12mole percent or more N-vinylpyrrolidone.
 10. The solid phase extractioncartridge of claim 1 comprising from about 0.001 g to about 10 g ofcopolymer.
 11. The solid phase extraction cartridge of claim 10comprising from about 0.025 g to about 1 g of copolymer.
 12. The solidphase extraction cartridge of claim 1 further comprising one or moreporous retaining means adjacent to the polymer.
 13. The solid phaseextraction cartridge of claim 12 wherein at least one porous retainingmeans is a filter element.
 14. A microtiter well plate suitable forextraction comprising a water-wettable polymer packed inside anopen-ended container, said polymer being formed by copolymerizing atleast one hydrophobic monomer and at least one hydrophilic monomerhaving a hydrophilic to hydrophobic monomer ratio sufficient for thepolymer to be water-wettable and wherein the polymer comprises greaterthan at least 12 mole percent of hydrophilic monomer.
 15. The microtiterwell plate of claim 14 wherein the hydrophilic monomer is selected fromthe group consisting of 2-vinylpyridine, 3-vinylpyridine and4-vinylpyridine.
 16. The microtiter well plate of claim 14 wherein thehydrophilic monomer is N-vinylpyrrolidone.
 17. The microtiter well plateof claim 14 wherein the hydrophobic monomer comprises a phenyl group, aphenylene group or a straight chain or branched C₂-C₁₈-alkyl group. 18.The microtiter well plate of claim 17 wherein the hydrophobic monomer isstyrene or divinylbenzene.
 19. The microtiter well plate of claim 14wherein the polymer is a poly(divinylbenzene-co-N-vinylpyrrolidone)copolymer.
 20. The microtiter well plate of claim 19 wherein the polymercomprises about 12 mole percent or more N-vinylpyrrolidone.